Connectivity mapping is a well-known hypothesis generating and testing tool having successful application in the fields of operations research, computer networking and telecommunications. The undertaking and completion of the Human Genome Project, and the parallel development of very high throughput high-density DNA microarray technologies enabling rapid and simultaneous quantization of cellular mRNA expression levels, resulted in the generation of an enormous genetic database. At the same time, the search for new pharmaceutical actives via in silico methods such as molecular modeling and docking studies stimulated the generation of vast libraries of potential small molecule actives. The amount of information linking disease to genetic profile, genetic profile to drugs, and disease to drugs grew exponentially, and application of connectivity mapping as a hypothesis testing tool in the medicinal sciences ripened.
The general notion that functionality could be accurately determined for previously uncharacterized genes, and that potential targets of drug agents could be identified by mapping connections in a data base of gene expression profiles for drug-treated cells, was spearheaded in 2000 with publication of a seminal paper by T. R. Hughes et al. [“Functional discovery via a compendium of expression profiles” Cell 102, 109-126 (2000)], followed shortly thereafter with the launch of The Connectivity Map (C-map Project by Justin Lamb and researchers at MIT (“Connectivity Map: Gene Expression Signatures to Connect Small Molecules, Genes, and Disease”, Science, Vol. 313, 2006.) In 2006, Lamb's group began publishing a detailed synopsis of the mechanics of C-map construction and installments of the reference collection of gene expression profiles used to create the first generation C-map and the initiation of an ongoing large scale community C-map project.
The basic paradigm of predicting novel relationships between disease, disease phenotype, and drugs employed to modify the disease phenotype, by comparison to known relationships has been practiced for centuries as an intuitive science by medical clinicians. Modern connectivity mapping, with its rigorous mathematical underpinnings and aided by modern computational power, has resulted in confirmed medical successes with identification of new agents for the treatment of various diseases including cancer. Nonetheless, certain limiting presumptions challenge application of C-map with respect to diseases of polygenic origin or syndromic conditions characterized by diverse and often apparently unrelated cellular phenotypic manifestations. According to Lamb, the challenge to constructing a useful C-map is in the selection of input reference data which permit generation of clinically salient and useful output upon query. For the drug-related C-map of Lamb, strong associations comprise the reference associations, and strong associations are the desired output identified as hits.
Noting the benefit of high-throughput, high density profiling platforms which permit automated amplification, labeling hybridization and scanning of 96 samples in parallel a day, Lamb nonetheless cautioned: “[e]ven this much firepower is insufficient to enable the analysis of every one of the estimated 200 different cell types exposed to every known perturbagen at every possible concentration for every possible duration . . . compromises are therefore required” (page 54, column 3, last paragraph). Hence, Lamb confined his C-map to data from a very small number of established cell lines. This leads to heightened potential for in vitro to in vivo mismatch, and limits information to the context of a particular cell line. Selection of cell line, therefore, may be critical to the utility of a resulting C-map.
Lamb stresses that particular difficulty is encountered if reference connections are extremely sensitive and at the same time difficult to detect (weak), and Lamb adopted compromises aimed at minimizing numerous, diffuse associations. Since the regulatory scheme for drug products requires high degrees of specificity between a purported drug agent and disease state, and modulation of disease by impacting a single protein with a minimum of tangential associations is desired in development of pharmaceutical actives, the Lamb C-map is well-suited for screening for potential pharmaceutical agents despite the Lamb compromises.
The connectivity mapping protocols of Lamb would not be predicted, therefore, to have utility for hypothesis testing/generating in the field of cosmetics. Cosmetic formulators seek agents or compositions of agents capable of modulating multiple targets and having effects across complex phenotypes and conditions. Further, the phenotypic impact of a cosmetic agent must be relatively low by definition, so that the agent avoids being subject to the regulatory scheme for pharmaceutical actives. Nonetheless, the impact must be perceptible to the consumer and preferably empirically confirmable by scientific methods. Gene transcription/expression profiles for cosmetic conditions are generally diffuse, comprising many genes with low to moderate fold differentials. Cosmetic agents, therefore, provide more diverse and less acute effects on cellular phenotype and generate the sort of associations expressly taught by Lamb as unsuitable for generating connectivity maps useful for confident hypothesis testing.
The present inventors surprisingly discovered that useful connectivity maps could be developed from cosmetic active—cellular phenotype—gene expression data associations in particular with respect to hair care cosmetics. Specifically, certain aspects of the present invention are based on the surprising discovery that selection of human cells such as fibroblasts, keratinocytes, melanocytes or dermal papilla cells are relevant cell lines and that data from such cells has resulted in construction of connectivity maps useful for hypothesis generating and testing relating to cosmetic agents in treatment of specific hair biology conditions such the appearance of gray hair, chronogenetic alopecia, senile alopecia, androgenetic alopecia, loss of hair diameter or hair breakage/fragile hair; for example, BJ fibroblasts are a better cell line than tert-keratinocytes for the identification of Monoamine Oxidase B (MAOB) inhibitors to improve hair growth. Melanocytes are cells of a better cell line for evaluating material to delay the appearance of gray hair, while a combination of cells appeared most suitable for other specific hair biology conditions. For example, a set of biological signatures were generated and combined from different cell lines and clinical data was generated from samples with multiple cell types to capture different aspect for hair growth and healthy fiber quality. Therefore, it could not be accurately predicted that data from one cell type (such as a fibroblast cell or a keratinocyte cell), or any combination thereof, could be used to construct a connectivity map effective for generating and testing hypotheses relating to cosmetic actives and genes associated with a specific hair biology condition.
Hair is a complex, multi-layered and dynamic system that provides a protective covering from elements and acts to disperse products from glands in acting as an interactive boundary between an organism and the environment. It is also vitally important to both individual health and self image. For example, a significant industry has developed to assist individuals with conditions of hair loss (alopecia) as well as to deal with excessive hair growth. In fact, a large array of hair conditions and disorders have been characterized and include alopecia, androgenic alopecia, alopecia greata, permanent alopecia, anagen growth state disorders, anagen effluvium, bulb disorders, bulge disorders, catagen and regression disorders, club hair, hirsutism, hypertrichosis, lanugo hair, miniaturization, telogen disorders, telogen effluvium, terminal hair, and vellus hair as non-limiting examples. FIG. 1 illustrates basic hair anatomy.
Due to the complexity of hair and its interaction with skin, a basic discussion of each is herein included. This discussion is necessary as various treatments of hair biology conditions include application of products or methods related to the hair itself or to the skin surrounding the hair or parts of the hair. For example, various hair treatments include methods and uses of products such as Rogaine®, Propecia®, hair transplants, hair electrolysis, and laser hair removal.
Though the intricacies of hair growth disorders is complex and requires additional research and breakthroughs, basic hair anatomy is well known, and has been previously described. For example a review of hair biology has been written by Ralf Paus and George Cotsarelis (see among other places, Paus R and Cotsarelis G. The Biology of Hair Follicles (Review Article). Mechanisms of Disease, Vol. 341 (7), 1999, pp. 491-497).
A hair contains a hair shaft that extends primarily out from the human skin surface, and having a distal portion recessing into the epidermis of the skin. Outlining the anatomy simplistically, within the skin is the hair follicle, bulb, and papilla. The hair shaft contains keratin. Within the skin, blood vessels nourish cells in the hair bulb and cellular materials including hormones can be transferred via such networks of the vasculature. Hair color is also controlled largely by pigment cells producing melanin in the hair follicle.
More generally, hair follicles cover the vast majority of the body surface. There are approximately 5 million hair follicles on the body with 100,000 on the scalp, with a density of up to roughly 300 to 500 hairs per square centimeter on the scalp. The great significance of the hair follicle requires an outlining of additional follicle details. The hair follicle can be divided anatomically into multiple parts, including the bulb consisting of the dermal papilla and matrix, the suprabulbar area from the matrix to the insertion of the arrector pili muscle, the isthmus that extends from the insertion of the arrector pili muscle to the sebaceous gland, and the infundibulum that extends from the sebaceous gland to the follicular orifice. The lower portion of the hair follicle consists of multiple portions: the dermal papilla, matrix, hair shaft (consisting from inward to outward the medulla, cortex, and cuticle), inner root sheath (IRS) consisting of the inner root sheath cuticle, Huxley's layer, Henle's layer, and the outer root sheath. The base of the follicle is invaginated by the dermal papilla, which has a capillary loop that passes through the papilla. Signal transduction and communication between the dermal papilla and the matrix cells influence how long and how thick the hair shaft will grow. The melanocytes within the matrix also produce the pigment in the hair shaft.
As indicated earlier, the hair shaft contains keratin. As for the hair medulla, this is only partially keratinized and therefore appears amorphous and may not always be present. The hair cortex cells lose their nuclei during their upward growth and do not contain any keratohyaline or trichohyaline granules. The keratin of the cortex is hard in contrast to the IRS and epidermis, which are soft. The cuticle is firmly anchored to the IRS cuticle. The cuticle of the IRS consists of a single layer of flattened overlapping cells that point downward and interlock tightly with the upward angled cells of the hair shaft cuticle. Huxley's layer is composed of two cell layers, whereas the outer Henle's layer is only one cell thick. Just before the isthmus, the IRS becomes fully keratinized but disintegrates at the level of the isthmus. Although the IRS is not present in the emerging hair shaft, the IRS serves as a strong scaffold in the lower portion of the hair follicle.
Returning once again to the hair follicle, the hair follicle is significant in hair development and cycling of hair follicles involves three main stages, including anagen, catagen, and telogen. The anagen phase is known as the growth phase, and a hair can spend several years in this phase. The catagen phase is a transitional phase occurring over a few weeks, with hair growth slowing and the follicle shrinking. The telogen phase is a resting phase where, over months, hair growth stops and the old hair detaches from the follicle; a new hair begins the growth phase and pushes the old hair out.
As indicated, an intricate relationship exists between hair and skin. Regarding the skin, the skin comprises three principal layers, the epidermis, the dermis, and a layer of subcutaneous fat. The majority of cells in the epidermis are keratinocytes that produce a family of proteins called keratins. Keratins contribute to the strength of the epidermis. The epidermis itself may be divided into multiple layers with the outermost layer referred to as the stratum corneum, and the innermost layer referred to as the basal layer. All epidermal cells originate from the basal layer and undergo a process known as differentiation as they gradually displace outward to the stratum corneum, where they fuse into squamous sheets and are eventually shed. In healthy, normal skin, the rate of production equals the rate of shedding (desquamation).
The differentiating epidermal cells form distinct though naturally blended layers. As the cells displace outward, they flatten and become joined by spiny processes forming the stratum spinosum, or spinous layer. The cells manufacture specialized fats called sphingolipids, and begin to express keratins associated with terminal differentiation. As keratin is produced, it is incorporated into the cellular matrix, strengthening the skin and providing structural support to the outer layers. As the cells migrate further outward and develop characteristic granules that contain proteins which contribute to the aggregation of keratins; they now form part of the granular layer. Cells lose their nuclei in the outer part of this layer, and the granules release their contents contributing to cornification. Vesicles containing lipids discharge into the spaces between the cells, creating a barrier structure that has been suggested to function like bricks (cells) and mortar (lipids). As the cells rise into the outermost layer of the epidermis—the stratum corneum, sometimes called the horny layer or the cornified layer—they take the form of flattened discs, tightly packed together. These flattened cells, called corneocytes, are effectively dead. The lipids of the epidermis play an important role in maintaining skin health, as they help the stratum corneum to regulate water loss while providing a virtually impermeable hydrophobic barrier to the environment. Fully mature keratinocytes function to protect the skin from UV light damage, and help effectuate immune responses to environmental stimuli.
The dermis, which lies just beneath the epidermis, is composed largely of the protein collagen. Most of the collagen is organized in bundles which run horizontally through the dermis and which are buried in a jelly-like material called the ground substance. Collagen accounts for up to 75% of the weight of the dermis, and is responsible for the resilience and elasticity of skin. The collagen bundles are held together by elastic fibers running through the dermis. The fibers are comprised of a protein called elastin, and make up less than 5% of the weight of the dermis. Fibroblasts function to synthesize collagen and the dermis ground substance, including components glycoproteins and glycosaminoglycans such as hyaluronic acid (which is able to bind water). The junction between the epidermis and the dermis is not straight but undulates—more markedly so in some areas of the body than others. A series of finger-like structures called rete pegs project up from the dermis, and similar structures project down from the epidermis. These projections increase the area of contact between the layers of skin, and help to prevent the epidermis from being sheared off. As skin ages, the projections get smaller and flatter. Networks of tiny blood vessels run through the rete pegs, bringing nutrients, vitamins and oxygen to the epidermis, although the epidermis itself is avascular and nourished by diffusion from the rete pegs. The dermis also contains the pilobaceous units comprising hair follicles and sebaceous glands, apocrine and eccrine sweat glands, lymphatic vessels, nerves, and various sensory structures, including the mechano-sensing Pacinian and Meissner's corpuscles.
Beneath the dermis lies the hypodermis, which comprises subcutaneous fat that cushions the dermis from underlying tissues such as muscle and bones. The fat is contained in adipose cells embedded in a connective tissue matrix. This layer may also house the hair follicles when they are in the growing phase.
Thus, skin is a multilayered complex organ comprising a wide variety of cellular types and structures, including epidermal and dermal connective tissue with blood and lymphatic vessels, the pilosebaceous units, glands, nerves, various sensory structures, the hypodermal adipose tissue, and the elastic fascia beneath the hypodermis. In turn, these structures are composed of a number of different cell types including keratinocytes, melanocytes, neuroendocrine Merkel cells, sebocytes, fibroblasts, endothelial cells, pericytes, neurons, adipocytes, myocytes and resident immunocytes including Langerhans cells, other dendritic cells, T cells and mast cells. Two of the main cell lineages in the skin are epithelial cells, which in general form the linings of the body and the parenchyma of many organs and glands, and mesenchymal cells, which form connective tissue, blood vessels and muscle. Dermal fibroblasts are mesenchymal cells, and keratinocytes are epithelial cells, which comprise most of the structure of the epidermis.
Thus hair and skin are intricate components that work in a complex manner to regulate hair health. As stated, there are a significant number of hair disorders, and there are many hair care products available to consumers which are directed to improving the health and/or physical appearance of hair. Despite current treatments, an ongoing need exists to identify cosmetic agents that can provide new or improved benefits to hair. There is also a need to identify additional cosmetic agents that provide similar or improved benefits as compared to existing products but which are easier to formulate, produce, and/or market.
Successful identification of hair-related cosmetic agents has proven to be difficult due to the multi-cellular, multi-factorial processes that occur in and around hair. In addition, many desirable cosmetic agents may comprise a mixture of compounds with effects and interactions that may not be fully understood. This is often the case with a botanical or other natural extract that may affect many cellular/pathways. An additional challenge for cosmetic formulators is that cosmetics must be very safe and adverse effects generally are not acceptable. Further, while much is known about hair biology, there is much that is still poorly understood or unknown. Conventional in vitro studies of biological responses to potential cosmetic agents involve testing hundreds or thousands of potential agents in various cell types before an agent that gives the desired result can be identified and moved into a next stage of testing. However, such studies can be hindered by the complex or weakly detectable responses typically induced and/or caused by cosmetic agents. Such weak responses arise, in part, due to the great number of genes and gene products involved, and cosmetic agents may affect multiple genes in multiple ways. Moreover, the degree of bioactivity of cosmetic agents may differ for each gene and be difficult to quantify.
The value of a connectivity map approach to discover functional connections among cosmetic phenotypes of hair biology, gene expression perturbation, and cosmetic agent action is counter-indicated by the progenitors of the drug-based C-map. The relevant phenotypes are very complex, the gene expression perturbations are numerous and weak, and cosmetic agent action is likewise diffuse and by definition, relatively weak. It has thus far been unclear whether statistically valid data could be generated from cosmetic C-maps and whether a cell line existed to provide salient or detectable cosmetic data.
Surprisingly, the present inventors have provided a C-map approach that is generalizable and biologically relevant for identification of potential cosmetic actives, and demonstrate that the C-map concept is viable by (as a non-limiting example) use of benchmark cosmetic actives to query the reference data and by identification of new cosmetic actives.